Patent Application
20250171490
The invention relates to a novel steroid for use against arteriosclerosis and/or atherosclerosis as well as pharmaceutical compositions and medical uses thereof.
Atherosclerosis is an inflammatory disease associated with hardening of the arteries, which can cause a variety of consequences including chronic kidney disease (CKD), heart attacks, stroke or thrombosis. According to WHO (World Health Organization), atherosclerosis is a considerable cause of mortality and morbidity worldwide (31%). The progression of atherosclerosis is linked to the development of calcification of heart valves called calcific aortic valve disease (CAVD). It is demonstrated by multiple studies that CAVD is accompanied by lipid accumulation, inflammation, and calcification resulting within the heart valvular tissue [Lusis A J, et al. 2004; Mohler E R, 2004; Speer M Y, et al. 2004; Mohler E R, 2001; Chester A. 2011]. Nowadays, CAVD is a cardinal problem as pharmacology drugs against aortic valves calcification are not available [Yutzey K E, et al. 2014].
Békési et al. have found that sex steroids and cortisol significantly reduced the release of superoxide anion from human neutrophils [Békési et al. 2000].
Brancaleone V. et al. evaluated H2S biosynthesis in rat isolated aortic rings following androgen receptor stimulation and found that H2S biosynthesis in the rat aorta is modulated by androgen hormones but is not triggered by female hormones 17-beta-ostradiol or progesterone. They conclude that androgens can exert protective actions on cardiovascular and metabolic functions by triggering a variety of beneficial effects mediated by H2S [Brancaleone et al. 2015].
Naftolin F et al. have shown that estradiol-induced neural cell adhesion molecule (nCAM) sialylases are present in vascular endothelial cells and that pretreatment of human arterial endothelial cells with estradiol, testosterone, dehydroepiandrosterone and dihydrotestosterone all induced sialylation of endothelial cells, reduced the capture of monocytes and are protective against atherogenesis and its sequellae [Naftolin F et al. 2016].
Dehydroepiandrosterone (DHEA) was found in early studies already in the '90s to inhibit iron dependent lipid peroxidation and to enhance the superoxide dismutase generation in hepatic mitochondria, and that it is preventive in the development of atherosclerosis [Schauer J E et al. 1990; Aragno M. et al. 1993].
Magyar et al. have studied the role of sulphatation-desulphatation of DHEA/DHEAS (dehydroepiandrosterone sulphate) in Sprague-Dawley adult male rats in vivo by steroid sulphotransferases and sulphatases and found that DHEAS had a more pronounced effect than DHEA on total scavenger capacity (TSC) [Magyar et al. 2011].
Hochberg R B et al. studied and reviewed already in the early '90s the role of steroidal fatty acid esters and reported that androsterone esters have been found in human breast tumors and breast cyst fluid [Hochberg et al. 1991].
A decade later Loria, Roger M. suggested that 5-androstene-3beta, 17alpha-diol and possibly its esters are inhibitors of tumor growth. Otherwise, the literature of such ester derivatives is sparse [Loria, Roger M., US 2001/0014675 A1].
Song, Ching et al. [Song et al. 2001] teaches auto-oxidized cholesterol sulfates are antagonistic ligands of liver X receptors and suggest that since LXR agonists can counteract the activity of these antagonists they may have therapeutic potential against atherosclerosis. However, the authors have found that non-sulfated forms of these compounds as well as many other steroid sulfates, including sulfate of 5-androsten-3B, 17β-diol, had no significant effect, contrary to 5a,6α-epoxycholesterol.
It is to be mentioned that the administration of steroids is counter-indicated in human medicine because, besides the antioxidant effect, they also have strong endocrinological effect which is clearly undesired in case of an anti-atherogenic medicament.
Moreover, the present inventors have found that in animal experiments neither DHEA nor DHEAS (dehydroepiandrosterone sulphate) had actual positive effect against atherosclerosis whereas both compounds proved to be toxic and induced mortality.
The present inventors have unexpectedly found that a narrow range of alkanoyl esters of DHEAS prevent and treat atherosclerotic plaque formation and calcification in ApoE/mice fed with atherogenic diet. Therefore, DHEA-derived steroid represents a novel potential treatment preventing and/or reversing atherosclerosis and valvular calcification.
The invention relates to compounds according to formula (I),
Preferably the C7-C13 alkyl is n-alkyl, i.e. a linear alkyl.
Preferably, R1 is C9-C13 alkyl.
Particularly preferably, R1 is C7-C1 alkyl.
Particularly preferably, R1 is C11-C13 alkyl.
In a further embodiment, R1 is a C8-C12 or a C9-C12 alkyl.
In a further embodiment, R1 is a C8-C11 or a C9-C1 alkyl.
In an embodiment, R1 is C10-C12 alkyl.
More particularly preferably, R1 is C11 alkyl.
In a preferred embodiment, R1 is any alkyl defined above. Preferably R1 is n-alkyl.
Highly preferably, R1 is C11 n-alkyl, i.e. the compound is 17-lauroyloxy-5-androstene-3-sulphate.
Preferably the salt is sodium salt or potassium salt, in particular sodium salt.
Preferably the alkyl is n-alkyl, i.e. a linear alkyl.
Preferably the salt is sodium salt. Preferably the solvate is water solvate.
In an embodiment the configuration at carbon 3 is 3β.
In an embodiment the configuration at carbon 17 is 17β.
In a preferred embodiment the configuration is 3β,17β.
In an embodiment the configuration at carbon 3 is 3α.
In an embodiment the configuration at carbon 17 is 17α.
In an embodiment any of the compounds of general formula (I) has general formula (Is1)
The invention relates to any of the compounds according to the invention for use in the treatment of arteriosclerosis, in particular atherosclerosis in a subject. The subject is a vertebrate having a blood circulation system (cardiovascular system or the vascular system), e.g. a mammalian subject as defined herein. The mammalian subject is preferably a human subject.
The invention relates to any of the compounds according to the invention for use in the prevention or reducing the risk of atherosclerosis.
The invention relates to any of the compounds according to the invention for use in the prevention of or in reducing the formation of or in alleviating the formation of or in reducing the risk of atherosclerotic lesions in arteries.
The invention relates to any of the compounds according to the invention for use in the prevention of or in reducing the formation of or in alleviating the formation of or in reducing the risk of atherosclerotic plaques in arteries.
The invention relates to any of the compounds according to the invention for use in the treatment of atherosclerosis.
The invention relates to any of the compounds according to the invention for use in the therapeutic treatment of atherosclerosis. The invention relates to any of the compounds according to the invention for use in the reversion of atherosclerosis or reversion of the formation of atherosclerotic lesions in arteries.
The invention relates to any of the compounds according to the invention for use in the reversion of atherosclerosis or of the formation of atherosclerotic plaques in arteries.
Preferably, the invention relates to any of the compounds provided hereinabove for use in the prevention of the onset of or formation of or in reducing the risk of any of the following conditions (including one or more of the following conditions):
Preferably, the invention relates to any of the compounds provided hereinabove for use in the treatment of or reversing the formation of any of the following conditions (including one or more of the following conditions):
The invention also relates to any of the compounds provided hereinabove for use in reducing the amount of reactive oxygen species (ROS) and/or inhibiting ROS production; thereby reducing oxidative stress and preventing the onset of or alleviating oxidative stress diseases.
Preferably, in the therapeutic method, preferably preventive method, the compound is selected from the group consisting of compounds according to formula (I), wherein R1 is C7-C13 alkyl; or any of the preferred compounds or any compound for use in treatment or prevention.
The invention also relates to a pharmaceutical composition or medicament comprising the compound of the invention, wherein said compound is selected from the group consisting of compounds according to formula (I), wherein R1 is C7-C13 alkyl; or any of the preferred compounds, and any pharmaceutically acceptable salt thereof, preferably its sodium salt,
Preferably the C7-C13 alkyl is n-alkyl, i.e. a linear alkyl.
Preferably, R1 is C9-C13 alkyl.
Particularly preferably, R1 is C7-C11 alkyl.
Particularly preferably, R1 is C11-C13 alkyl.
In a further embodiment, R1 is a C8-C12 or a C9-C12 alkyl.
In a further embodiment, R1 is a C8-C1 or a C9-C11 alkyl.
In an embodiment, R1 is C10-C12 alkyl.
More particularly preferably, R1 is C11 alkyl.
In a preferred embodiment, R1 is any alkyl defined above. Preferably R1 is n-alkyl.
Highly preferably, R1 is C11 n-alkyl, i.e. the compound is 17-lauroyloxy-5-androstene-3-sulphate.
Preferably the salt is sodium salt. Preferably the salt is potassium salt.
Preferably the alkyl is n-alkyl, i.e. a linear alkyl.
Preferably the salt is sodium salt. Preferably the solvate is water solvate.
In an embodiment the configuration at carbon 3 is 3β.
In an embodiment the configuration at carbon 17 is 17β.
In a preferred embodiment the configuration is 3β,17β.
In an embodiment the configuration at carbon 3 is 3α.
In an embodiment the configuration at carbon 17 is 17α.
The invention relates to a pharmaceutical composition comprising the compound according to the invention, said composition also comprising a pharmaceutically acceptable carrier or excipient. Preferably the compound according to the invention is a compound for use as defined herein; preferably a compound as defined above in the Compound section.
The invention relates to a pharmaceutical composition for use in a subject according to the invention for prevention against a condition as taught herein, said composition also comprising a pharmaceutically acceptable carrier or excipient.
Pharmaceutically acceptable carriers or excipients are known for a person skilled in the art.
Preferably the pharmaceutical composition is for or is suitable for enteral, e.g. oral or parenteral, e.g. intravenous, or topical administration into a patient. Oral administration is preferred.
Said oral pharmaceutical compositions may be formulated e.g. as pills, tablets, tabs, coated tablets, film tablets, capsules, powders, granulates, sustained-release formulations, suspensions or drops.
Intravenous pharmaceutical compositions may be formulated e.g. as liquid solution preparations for injections or infusion, and can be administered in any usual intravenous administration means, e.g. through an intravenous line (cannula) or by injections.
The pharmaceutical compositions may be formulated for topical administration, e.g. as drops, sprays, aerosols, ointments, creams, pastes, syrup, lotion or gels which may be useful for administration near to the site of the atherosclerotic lesion or plaque.
Thus, the pharmaceutical compositions may be formulated for systemic administration, like oral and intravenous compositions, or for local administration like topical compositions or other local administration compositions like sublingual, buccal, etc. administration.
Highly preferably the pharmaceutical composition is present in a form suitable for oral administration. The pharmaceutical composition in the form of tablets is most preferred.
In a preferred embodiment the pharmaceutical composition of the invention is formulated in a form for administration of a daily dose as defined below under Methods for treatment, preferably in one, two or three parts a day. Preferably the form is for oral administration.
In a preferred embodiment the administration form, in particular in human patients, comprises a dose of at least 0.5 mg, 1 mg, 2 mg. 5 mg, 10 mg, 15 mg, 20 mg or 30 mg.
In a preferred embodiment the administration form, in particular in human patients, comprises a dose of at most 500 mg, 200 mg, 150 mg or 100 mg, 80 mg, 50 mg or 40 mg.
In a preferred embodiment the administration form, in particular in human patients, comprises a dose between 0.5 and 500 mg, between 1 and 500 mg, or between 5 and 200 mg, or in particular between 10 and 100 mg, or between 20 and 200 mg, or between 5 and 100 mg, or in more particular between 10 and 80 mg, or 20 and 150 mg, or 1 to 30 mg or 1 to 40 mg or 1 to 60 mg, or 5 to 20 mg, or 5 to 40 mg or 5 to 60 mg or 10 to 40 mg.
In another preferred embodiment, the administration dose for a human is 10 to 40 mg/day.
In another preferred embodiment, the administration dose for a human is between 0.01 mg/kg and 5 mg/kg or between 0.01 mg/kg and 1 mg/kg, preferably between 0.01 mg/kg and 0.5 mg/kg, in particular between 0.01 mg/kg and 0.4 mg/kg, or between 0.01 mg/kg and 0.3 mg/kg or between 0.01 mg/kg and 0.05 mg/kg, or between 0.05 mg/kg and 0.5 mg/kg. In these doses, per kg refers to per kilogram of body weight.
In another preferred embodiment, the administration dose for a mouse is a dose between 1 μg/day and 200 μg/day, in particular 4 μg/day to 100 μg/day or 4 μg/day to 20 μg/day or 20 μg/day to 100 μg/day.
In another preferred embodiment, the administration dose for a mouse is a dose between 0.1 mg/kg and 10 mg/kg, in particular between 0.1 mg/kg and 5 mg/kg, or between 0.1 mg/kg and 1 mg/kg, or between 0.5 mg/kg and 5 mg/kg or between 1 mg/kg and 5 mg/kg. In these doses, per kg refers to per kilogram of body weight.
In an embodiment, the dose is a dose corresponding to any of the above doses calculated for another animal under appropriate guidelines (see e.g. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (2005). Guidance for Industry—Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers).
The invention also relates to methods for treatment, wherein the pharmaceutical composition is administered to a subject or patient in an effective dose.
According to the invention the administration routes listed above in connection with the pharmaceutical compositions can be used in the methods for treatment of the invention for administration of the composition to a subject. The subject is a vertebrate having a blood circulation system (cardiovascular system or the vascular system), e.g. a mammalian or human subject as defined herein.
In a preferred embodiment the daily dose of administration, in particular in human patients, is at least 0.5 mg, 1 mg/day, 5 mg/day, 10 mg/day, 20 mg/day or 30 mg/day.
In a preferred embodiment the daily dose of administration, in particular in human patients, is at most 500 mg/day, 200 mg/day, 150 mg/day or 100 mg/day or 80 mg/day.
In a preferred embodiment the daily dose of administration, in particular in human patients, is between 0.5 and 500 mg/day, between 1 and 500 mg/day, or between 5 and 200 mg/day, or in particular between 10 and 100 mg/day, or between 20 and 200 mg/day, or between 5 and 100 mg/day, or in more particular between 10 and 80 mg/day, or 20 and 150 mg/day, or 1 to 30 mg or 1 to 40 mg or 1 to 60 mg, or 5 to 20 mg, or 5 to 40 mg or 5 to 60 mg or 10 to 40 mg.
In another preferred embodiment, the administration dose for a human is 10 to 40 mg/day.
In another preferred embodiment, the administration dose for a human is between 0.01 mg/kg and 5 mg/kg or between 0.01 mg/kg and 1 mg/kg, preferably between 0.01 mg/kg and 0.5 mg/kg, in particular between 0.01 mg/kg and 0.4 mg/kg, or between 0.01 mg/kg and 0.3 mg/kg or between 0.01 mg/kg and 0.05 mg/kg, or between 0.05 mg/kg and 0.5 mg/kg. In these doses, per kg refers to per kilogram of body weight.
In another preferred embodiment, the administration dose for a mouse is a dose between 1 μg/day and 200 μg/day, in particular 4 μg/day to 100 μg/day or 4 μg/day to 20 μg/day or 20 μg/day to 100 μg/day.
In another preferred embodiment, the administration dose for a mouse is a dose between 0.1 mg/kg and 10 mg/kg, in particular between 0.1 mg/kg and 5 mg/kg, or between 0.1 mg/kg and 1 mg/kg, or between 0.5 mg/kg and 5 mg/kg or between 1 mg/kg and 5 mg/kg. In these doses, per kg refers to per kilogram of body weight.
In an embodiment, the dose is a dose corresponding to any of the above doses calculated for another animal under appropriate guidelines (see e.g. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (2005). Guidance for Industry—Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers).
In an embodiment the effective dose means the administration of an amount, wherein the level of H2S is detectably or effectively elevated in the subject or patient.
The invention relates to a method for the treatment of atherosclerosis.
In the method of the invention, the compound of the invention is administered as disclosed herein to a subject, e.g. to a mammalian subject, preferably a human subject.
In an embodiment the method for treatment is a method for prevention or reducing the risk of atherosclerosis, or for prevention of or for reducing the formation of or for alleviating the formation of or for reducing the risk of atherosclerotic lesions or plaques in arteries.
In an embodiment the method for treatment is a method for the reversion of atherosclerosis or of the formation of atherosclerotic lesions or plaques in arteries.
Preferably, the invention relates to the method of treatment as taught herein, wherein said compound or composition is administered in the prevention of onset or formation of or in reducing the risk of any of the conditions listed above in the Compounds section or
In an embodiment the mammalian or human subject is diagnosed with atherosclerosis before said method of treatment is started.
In an embodiment the mammalian or human subject is diagnosed as a subject at risk of atherosclerosis before said method of treatment is started.
Such diagnostic methods are well known to a person skilled in the art as taught hereinbelow.
The invention also relates to a process for the preparation of a compound of the invention, said method comprising the steps of
In a preferred embodiment R1 is C7-C13 alkyl. Particularly preferably, R1 is C7-Cn alkyl. Particularly preferably, R1 is C11-C13 alkyl.
Preferably R1 is n-alkyl.
In another preferred embodiment, R1 is C9-C13 alkyl.
In a further embodiment, R1 is a C8-C12 or a C9-C12 alkyl.
In a further embodiment, R1 is a C8-C11 or a C9-C11 alkyl.
In an embodiment, R1 is C10-C12 alkyl.
More particularly preferably, R1 is C11 alkyl.
In a preferred embodiment, R1 is any alkyl defined above. Preferably R1 is n-alkyl.
In a preferred embodiment, in acylation step III a C10-C14-alkanoyloxy-halogenide is used and thereby in step V of the process 17-C10-C14-alkanoyloxy-5-androstene-3-sulphate is obtained.
In a particularly preferred embodiment, in acylation step III a C8-C12-alkanoyloxy-halogenide is used and thereby in step V of the process 17-C8-C12-alkanoyloxy-5-androstene-3-sulphate is obtained. In a particularly preferred embodiment, in acylation step III a C12-C14-alkanoyloxy-halogenide is used and thereby in step V of the process 17-C12-C14-alkanoyloxy-5-androstene-3-sulphate is obtained.
In a highly preferred embodiment, in acylation step III a C12-alkanoyloxy-halogenide is used and thereby in step V of the process 17-C12-alkanoyloxy-5-androstene-3-sulphate is obtained.
In an embodiment acylation step I is carried out by reacting the 3-hydroxy-17-oxo-5-androstene with acetic anhydride under appropriate conditions, e.g. in an aprotic apolar solvent, e.g. in dichloromethane, preferably at temperature T=0° C., preferably for 2 to 4 hours.
In an embodiment reduction step II is carried out by reacting the 3-acetoxy-17-oxo-5-androstene with a reducing agent, e.g. a hydride, in particular NaBH4, under appropriate conditions, e.g. in a protic polar solvent, e.g. in methanol, preferably at temperature T<0° C., more preferably at −10° C. <T<0° C., even more preferably at −8° C. <T<−2° C., preferably for 1 to 5 hours, preferably for 2 to 3 hours.
In an embodiment acylation step III is carried out by reacting the 3-acetoxy-17-hydroxy-5-androstene with the respective C8-C14-alkanoyloxy-halogenide, preferably C8-C14-alkanoyloxy-chloride, under appropriate conditions, e.g. in an aprotic solvent, preferably in a mixture of an apolar and an aromatic solvent, preferably in a mixture of dichloromethane and pyridine, preferably at room temperature or at a temperature of 20° C. <T<30° C., preferably for less than one day, or 2 to 24 hours, preferably 4 to 16 hours.
In an embodiment hydrolysis step IV is carried out by hydrolyzing the 3-acetoxy-17-C8-C14-alkanoyloxy-5-androstene under basic conditions, preferably by using an alkali hydroxide, e.g. sodium hydroxide in a protic polar solvent, e.g. in a mixture of methanol and water, at a temperature of 10° C. <T<30° C., preferably at a temperature of 15° C. <T<25° C. preferably for less than one day, or 4 to 24 hours, preferably 6 to 20 hours.
In an embodiment sulphation step V is carried out by sulphating the 3-hydroxy-17-C8-C14-alkanoyloxy-5-androstene with a sulphating agent, preferably chlorosulfonic acid, preferably in a mixture of an apolar and an aromatic solvent, preferably in a mixture of dichloromethane and pyridine, preferably at a temperature of T<0° C., more preferably at −10° C. <T<0° C., even more preferably at −8° C. <T<−2° C., preferably for 1 to 12 hours, preferably for 2 to 8 hours.
In a preferred embodiment appropriate salt is formed by using a metal salt forming reagent, preferably an alkali metal salt, e.g. in water comprising preferably the corresponding alkali carbonate or hydrogen carbonate, preferably by adding the metal salt forming reagent to the sulphating reaction mixture once the sulphating reaction is allowed to proceed for a sufficient time. Optionally salt is formed in a multiple phase, e.g. three-phase reaction, which is diluted by an apolar volatile solvent, e.g. diethyl ether and the salt is filtered out and purified, optionally recrystallized.
In a preferred embodiment the process of the invention comprises the steps of
“Arteriosclerosis” is a condition wherein the walls of arteries are thickened, hardened, and lost their elasticity. Arteriosclerosis is considered herein as a disease.
“Atherosclerosis” is a specific type of arteriosclerosis wherein abnormal material is accumulated in the inner layer of the wall of an artery, which forms “atherosclerotic lesions”, which are made up of fats, cholesterol and other substances in and on the walls of arteries and restrict blood flow.
“Atherosclerotic lesions” are abnormalities in the inner side of the arterial wall and have several types (types I to VI) based on their size, composition and severity. Less developed atherosclerotic lesions do not cause symptoms.
“Atherosclerotic plaques” or atheromas are abnormal accumulation of material in the inner layer of the wall of an artery, in particular atherosclerotic lesions which comprise extracellular lipids which are confluent, and which potentially cause symptoms in the subject affected by atherosclerosis.
A “subject” as used herein is an individual of an animal species having blood circulation and capable of developing arteriosclerosis or atherosclerosis, preferably a vertebrate, more preferably a mammalian or avian species, in particular a mammalian species, highly preferably the individual is a primate, a hominid or a human.
A “patient” is a subject who is or intended to be under medical or veterinarian observation, supervision, diagnosis or treatment.
A “treatment” refers to a process or method of administration of a pharmaceutical composition or medicament to a subject (or patient), wherein the subject or patient is under medical or veterinarian aid with the object of improving the subject's or patient's condition or restoring his/her health, either directly or indirectly or maintaining it in a healthy or stabilized state to avoid worsening. Treatment of the subject as used herein include restoring or maintaining normal function of an organ or tissue, preferably at least partly restoring or maintaining health (medical or veterinarian treatment). Treatment typically refers to the administration of an effective amount of a compound or composition described herein. Treatment may relate to or include medical or veterinarian treatment and cosmetic treatment, in particular medical or veterinarian treatment.
A “therapeutic treatment” (or therapy) is a treatment with the object of improving the subject's or patient's condition or restoring his/her health. For example, as used herein, the therapy includes reversion of atherosclerosis or reversion of the formation of atherosclerotic lesions in arteries.
A “preventive treatment” (or prevention) is a treatment with the object of preventing or avoiding the onset of or worsening of a disease or condition. For example, as used herein, prevention includes reducing the formation of or alleviating the formation of or reducing the risk of atherosclerotic lesions in arteries.
A “pharmaceutical composition” or “composition” of the invention is a composition of matter which comprises at least one biologically active substance of the invention suitable for increasing H2S level in the subject and thereby preventing the formation of atherosclerotic lesions or plaques. The treatment includes administration of the substance in an effective amount. Compositions may also comprise further biologically active substances useful e.g. in a combination therapy. Furthermore, the compositions may comprise pharmaceutically acceptable carriers and/or excipients, including formulation agents, etc., which are well known in the art. A medicament is a “pharmaceutical composition” marketed or to be marketed under a marketing authorization issued or to be issued by a respective authority in the geographic area given.
The terms “effective amount” and “therapeutically effective amount” are intended to qualify the amount of a therapeutic agent required to relieve or prevent to some extent one or more of the symptoms of a condition.
As used herein, the term “alkyl” alone or in combinations means a straight or branched-chain saturated hydro-carbon group of a length as given by the number of carbon atom. In a broader sense the alkyl may comprise substituents, e.g. halogen substituents. In a narrower sense the alkyl does not comprise a substituent.
As used herein, the term “alkanoyl” refers to a group which contains a double-bonded oxygen atom and an alkyl group having the formula R—C═O or R—(CO)—, wherein R represents an alkyl group that is linked to the carbon atom of the group by a single bond and the carbon atom may be linked by a single covalent bond to another moiety of the organic molecule. In organic chemistry, the alkanoyl group is usually derived from a carboxylic acid.
When used herein, the terms “halogen” and “halo” include fluorine, chlorine, bromine and iodine, and fluoro, chloro, bromo and iodo, respectively.
“Sulphation” (or “sulfation”) means herein a process wherein a sulphate ester is formed in an organic molecule.
As used herein, the term “solvate” is a crystal form containing the compound of the invention or a pharmaceutically acceptable salt thereof and either a stoichiometric or a non-stoichiometric amount of a solvent, by way of example, water. Reference to a compound of the invention includes any physical form of that compound, unless a particular form, salt or solvate thereof is specified.
The term “comprises” or “comprising” or “including” are to be construed herein as having a non-exhaustive meaning and allow the addition or involvement of further features or method steps or components to anything which comprises the listed features or method steps or components. “Comprising” can be substituted by “including” if the practice of a given language variant so requires or can be limited to “consisting essentially of” if other members or components are not essential to reduce the invention to practice.
The indefinite articles “a” or “an” may refer either to singular or plural if context allows.
Arteriosclerosis and atherosclerosis are diseases wherein the walls of arteries are thickened, hardened, and lost their elasticity, and in atherosclerosis blood flow may be hindered. These conditions may lead to more serious diseases. The causes of arteriosclerosis and atherosclerosis appear to be lipid retention and modification, oxidation by free radicals (oxidative stress), which provoke chronic inflammation at susceptible sites in the walls. Initial lesions including fatty streaks in arteriosclerosis evolve into fibrous atherosclerotic plaques, which may become vulnerable to rupture, and thus may cause thrombosis or stenosis [Insull, W 2009]. Risk factors for atherosclerosis and its thrombotic complications include hypertension, cigarette smoking and diabetes mellitus, and the role of the immune system is inevitable, chronic inflammation being a risk factor in itself.
Type I lesion contains atherogenic lipoprotein which elicit an increase in macrophages and formation of scattered macrophage foam cells. Type II lesion, also called as fatty streaks are formed by layers of macrophage foam cells and smooth muscle cells carrying lipids. Type III lesions contain scattered extracellular lipid droplets and particles that disrupt the coherence of some intimal smooth muscle cells. Type IV lesions also comprise extracellular lipids which are confluent and larger than in Type III lesions (the latter producing a transitional state between type II and type IV lesions). A type IV lesion and more serious lesions are also called atherosclerotic plaques or atheromas and are already potentially symptom-producing. Type V lesions usually have a lipid core and also contain layer(s) of fibrous connective tissue, whereas type VI lesions comprise fissure, hematoma, and thrombus (type VI lesion). Type V lesions have variants, and e.g. type Vb lesions are largely calcified and type Vc lesions consist mainly of fibrous connective tissue and little or no accumulated lipid or calcium. The plaque can burst, triggering a blood clot [Stary H C. et al. 1995].
The role of free radicals is investigated in connection with arteriosclerosis and atherosclerosis. Free radicals are molecules that contain an unpaired electron on their outer atomic orbital. These seek a pair for themselves, and accept an electron from their surroundings, thus a chain of production of new molecules with unpaired electrons begins. Free radicals are therefore highly reactive and are able to damage the surrounding biological substances, like proteins, carbohydrates, lipids, and nucleic acids. This leads to the development of free radical mediated diseases, like atherosclerosis.
Earlier research with the radical neutralizing, antioxidant, also known as scavenging effect of steroid hormones, have proven that a number of steroids, like sex hormones and cortisol are effective antioxidants in isolated human neutrophil granulocytes. It has also been shown in the art that these steroids, besides the antioxidant effect, also have strong endocrinological effect, thus their preventive administration in human medicine is out of the question.
The present inventors have started from a very weak androgen, dehydroepiandrosterone (DHEA) as initial molecule and surprisingly found that the binding of certain substituents to the 3rd and 17th carbon atom of the sterane frame bears antioxidant property. Besides preventing the formation of atherosclerotic lesions and plaques in an animal model, the DHEA-derived steroids according to the invention reverse the atherosclerotic lesions and plaques previously formed in the animals.
Thus, the DHEA-derived steroids of the invention represent a novel atheroprotective drug, usable for preventing the formation of lipid derivatives in the atherosclerotic lesions and plaques, and inhibiting and also reversing calcification of heart valves.
Using organic chemical methods, the present inventors placed a sulfate group in the 3rd position and a lauroyloxy hydrocarbon chain in the 17th position. Therefore, the present inventors created a set of novel steroid compounds, 17-alkyloxy-5-androstene-3-sulphates, which proved to be highly antioxidant in isolated human neutrophil granulocytes, without substantive endocrinological effect. In a preferred embodiment the 17 alkyl is n-alkyl.
The chemical name of a highly preferred new compound is: 17-lauroyloxy-5-androstene-3-sulphate (or 5-androstenediol-17-lauroyl-3-sulphate).
In particular embodiments the compound is selected from the group consisting of 17β-lauroyloxy-5-androstene-3β-sulphate, 17β-lauroyloxy-5-androstene-3α-sulphate, 17α-lauroyloxy-5-androstene-3β-sulphate and 17α-lauroyloxy-5-androstene-3α-sulphate. In a preferred embodiment the compound is 17β-lauroyloxy-5-androstene-3β-sulphate.
In a particular embodiment its sodium salt is used. The chemical formula is C31H51NaO6S.
The chemical steps were the following: 1: DHEA, 2:3-acetoxy-17-oxo-5-androstene, 3:3-acetoxy-17-hydroxy-5-androstene, 4:3-acetoxy-17-lauroyloxy-5-androstene, 5:3-hydroxy-17-lauroyloxy-5-androstene, 6:17-lauroyloxy-5-androstene-3-sulphate sodium salt. See below in detail.
A preferred compound has a hydrocarbon chain with 12 carbon atoms in the 17th position. We examined whether compounds with shorter (6 carbon atoms: 17-hexanoyloxy-5-androstene-3-sulphate sodium salt, see compound C6) or longer (16 carbon atoms: 17-palmitoyloxy-5-androstene-3-sulphate sodium salt, see compound C16) hydrocarbon chain have antiatherogenic effect. In animals treated with these compounds the size and the number of plaques were similar to the control mice (while a very small non-significant effect may have been present in case of compound C16). However, 17-lauroyloxy-5-androstene-3-sulphate sodium salt (see compound S2) proved to be highly efficient both in the prevention and reversal of atherosclerosis. This means, that the positive effect of the newly synthesized compound of the invention is bound to the range between 6 and 16 carbon atoms. Thus, the length of the chain of the alkanoyl group is at least 8 carbon atoms, i.e. R1 is C7 in general formula (I), i.e. n is 6 in general formula (I′) and (V), and at most 14 carbon atoms, i.e. R1 is C13 in general formula (I), i.e. n is 12 in general formula (I′) and (V).
Preferably, the length of the chain of the alkanoyl group is at least 10 carbon atoms, i.e. R1 is C9 in general formula (I), i.e. n is 8 in general formula (I′) and (V), and at most 14 carbon atoms, i.e. R1 is C13 in general formula (I), i.e. n is 12 in general formula (I′) and (V).
In a particular embodiment, the length of the chain of the alkanoyl group is at least 11 carbon atoms, i.e. R1 is C10 in general formula (I), i.e. n is 9 in general formula (I′) and (V), and at most 13 carbon atoms, i.e. R1 is C12 in general formula (I), i.e. n is 11 in general formula (I′) and (V).
In a highly preferred embodiment, the length of the chain of the alkanoyl group is 12 carbon atoms, i.e. R1 is C11 in general formula (I), i.e. n is 10 in general formula (I′) and (V).
The exemplary compound of the invention used in the experiments below is a 17-lauroyloxy-5-androstene-3-sulphate salt, preferably sodium salt.
The compound has the general formula (I′)
Thus, highly preferably R1 is (CH2)10—CH3.
In an embodiment the configuration at the 3rd carbon is S (3β).
In a further embodiment the configuration at the 3rd carbon is R (3α).
Preferably the configuration at the 3rd carbon is S (3β).
In the present specification, alternatively, the IUPAC numbering of the sterane can be used, however, positions 3 and 17 are the same by IUPAC and in the present numbering shown.
Upon preparation of the compound a mixture of beta and alpha configuration in respect of carbon 3 may be present. In an embodiment, the mixture may be a racemic mixture. In an embodiment either the beta or the alpha configuration in respect of carbon 3 may be in excess.
In a pharmaceutical composition mixture of beta and alpha configuration in respect of carbon 3 may be present. In an embodiment, the mixture may be a racemic mixture. In an embodiment either the beta or the alpha configuration in respect of carbon 3 may be in excess.
It is to be noted that the 3β configuration is a particular configuration.
For example, the configuration of the compound can be as shown on general formula (Is1′):
In an embodiment the configuration at the 17th carbon is R.
In a further embodiment the configuration at the 17th carbon is S.
In an embodiment the configuration at the 17th carbon is beta (17).
In a further embodiment the configuration at the 17th carbon is alpha (17α).
In an embodiment the stereochemistry of the compound is selected from the group consisting of 17β, 3β, 17β, 3α, 17α, 3β and 17α, 3α, preferably 17β,3β.
Upon preparation of the compound a mixture of beta and alpha configuration in respect of carbon 17 may be present. In an embodiment, the mixture may be a racemic mixture. In an embodiment either the beta or the alpha configuration in respect of carbon 17 may be in excess.
In a preferred embodiment the configuration is 3β,17β as shown by Formula (Is2).
For example, the configuration of the compound can be as shown on general formula (Is2):
In a pharmaceutical composition mixture of beta and alpha configuration in respect of carbon 17 may be present. In an embodiment, the mixture may be a racemic mixture. In an embodiment either the beta or the alpha configuration in respect of carbon 17 may be in excess.
It is to be noted that the 17β configuration (which is present in DHEA) is a particular configuration.
The newly synthesized compound(s) were tested ex vivo, on isolated human neutrophil granulocytes and also in in vivo animal model. The present inventors chose Apo-E deficient mice kept on a lipid rich diet that are used as atherosclerosis models. The treatment lasted eight weeks. Control animals were given plain drinking water and lipid rich food, while the treated group, kept on the same diet, were administered 100 micrograms per animal per day of the new compound synthesized by the present inventors. The compound was dissolved in the drinking water. At the end of the examination period, the size and number of atherosclerotic plaques developed in the aortic arch and the inner surface of the aorta, calcification of heart valves, and serum hydrogen sulfide levels were determined. The present inventors found that in treated animals the number and size of plaques and the extent of calcification was significantly lower, and the concentration of hydrogen sulfide, a strong antioxidant was significantly higher. The latter may serve as explanation of the mechanism of action.
In the further part of the present work, it was examined whether the compounds of the invention have positive therapeutic effect, too. In the above-mentioned animal model, the present inventors developed during eight weeks real atherosclerosis, and for an additional eight weeks animals were given S2 compound (100 micrograms per day per animal). Control mice got pure drinking water. At the end of experiment the size and number of sclerotic plaques, the calcification of heart valves and the amount of hydrogen sulfide was measured. In animals treated with the compound of the invention the size and number of plaques and the calcification were significantly less, the concentration of hydrogen sulfide was significantly higher than in control animals. This means the compounds of the invention have not only preventive, but also therapeutic effect.
Without being bound by theory, the present inventors have studied the possible mechanism behind the biological and medical effect.
In previous studies it was found that elevated H2S (I.) scavenged atherogenic free radical superoxide, (II.) limited the formation of pro-oxidant and pro-inflammatory lipid mediators and subsequent endothelial responses provoked by these species, (III.) prevented heart valves calcification by controlling osteoblast-like differentiation of valvular interstitial cells [Bekesi G, et al. 2000; Bekesi G, et al. 2004; Potor L, et al. 2018; Sikura K E, et al. 2019].
In a systematic study, it was found that neutrophils incubated with corticosterone and 18-hydroxy-deoxycorticosterone showed a significant reduction in superoxide production, whereas the present inventors found a significant enhancement in the presence of 11beta-hydroxyprogesterone. Furthermore, a non-significant decreasing trend was observed after incubation with cholesterol 3-sulphate and an increasing tendency using 11-hydroxyandrostenedione [Bekesi G, et al. 2004].
Hydrogen sulfide (H2S) is the newest member of the endogenous gaseous transmitter family along with nitric oxide and carbon monoxide [Wang R. 2002]. Potor et al. also showed that H2S exhibits an anti-atherosclerotic function in ApoE−/− mouse on a high-fat diet via inhibiting lipid-peroxidation. Furthermore, they presented that, exogenous H2S inhibited lipid oxidation of human atheroma and of human hemorrhage lesion [Potor L, et al. 2018]. Recently, Sikura and Potor et al. observed the inhibition of heart valves calcification by H2S in ApoE−/− mouse on a high-fat diet [Sikura K E, et al. 2019].
It has been found that an elevated level of H2S in the mice plasma due to DHEA-derived steroid treatment contribute to this effect. In view of this the present inventors wanted to clarify whether the basic chemical structures (DHEA and its sulphate DHEAS) of the compound of the invention have antiatherogenic property, and found that in the animal model these basic compounds did not diminish the development of atherosclerosis but elevated mortality rate of the animals (see e.g.
In conclusion, the present invention provides evidence that DHEA-derived steroid of the invention is potent preventive and therapeutic agent in arteriosclerosis as well as atherosclerosis and atherosclerosis-associated diseases.
The invention is useful in the treatment of a number of conditions and diseases.
Atherosclerosis is a pathological process in the arteries, in which the inside of an artery narrows due to the buildup of plaque. Arteries throughout the body can be affected, e.g., the coronary arteries, cerebral arteries, iliac and femoral arteries, and aorta, and it can cause various diseases, depending on which artery/arteries are affected [National Research Council (US) Committee on Diet and Health. (1989). Diet and Health: Implications for Reducing Chronic Disease Risk. National Academy Press, Washington D.C., 1989].
Lesions in the coronary arteries lead to CHD (coronary heart disease, also known as coronary artery disease=CAD), which is one of the most common and serious manifestations of atherosclerosis. The term coronary heart disease includes many syndromes, such as sudden cardiac death, myocardial infarction, stroke, heart attack, angina pectoris.
Sudden cardiac arrest or sudden cardiac death occurs when the electrical system to the heart malfunctions and becomes irregular. Blood flow to the body and to the brain is reduced, the affected person can lose consciousness and can die unless emergency treatment is begun immediately.
Myocardial infarction (or heart attack) occurs when blood flow decreases or stops to part of the heart, which causes ischemic necrosis of the myocardium. The necrotic tissue is replaced by connective tissue. The subsequent clinical outcome depends on the amount and location of the lost cardiac muscle.
The stenosis of the coronary arteries sometimes does not cause infarction, but it can cause ischemic pain, especially on exertion—this is called angina pectoris. This condition indicates the presence of severe lesions and high risk of myocardial infarction.
Stroke occurs when the flow of oxygen-rich blood to a portion of the brain is blocked. Without oxygen, the brain cells start to die, and the brain tissue becomes damaged. The two main types of stroke are ischemic (due to lack of blood), and hemorrhagic (due to bleeding). Thrombosis formed over an atherosclerotic plaque in a cerebral artery can decrease or interrupt blood flow to part of the brain, causing ischemic necrosis. The symptoms can be paralysis on the contralateral side, and disturbances of speech, vision, hearing, and memory.
Peripheral arterial disease (PAD) is an abnormal narrowing of arteries other than those that supply the heart or brain (e.g., abdominal aorta, iliac arteries, and femoral arteries). It occurs when atherosclerosis and its complications in the arteries produce temporary arterial insufficiency in the lower extremities or ischemic necrosis of the extremities. PAD includes several clinical syndromes of arterial insufficiency in the extremities, characterized by pain, inflammation, and ischemic damage to soft tissues from partial or complete occlusion of major arteries. PAD is often used interchangeably with lower extremity arterial disease (LEAD), when it affects the arteries of the lower extremities of the body.
Atherosclerosis can be associated with other diseases, as well.
Calcification can occur in atherosclerotic vascular lesions and in the aortic valve. Calcific aortic valve disease (CAVD) is a slow, progressive disorder. It has many forms from mild valve thickening, without obstruction of blood flow (aortic sclerosis) to severe calcification with impaired leaflet motion (aortic stenosis) [Lerman et al. 2015]. CAVD is the most common heart valve disease, and no medical treatment so far has been able to slow CAVD progression [Hulin et al. 2018].
Chronic kidney disease (CKD) can also be associated with atherosclerosis. The patients with CKD are at increased risk of atherosclerotic cardiovascular disease [Kon et al. 2014]. Accelerated atherosclerosis has been observed in early stages of renal dysfunction [Olechnowicz-Tietz et al. 2013]. A greater distinguishing feature of atherosclerotic cardiovascular disease in CKD is the severity of the disease, which is reflective of an increase in inflammatory mediators and vascular calcification secondary to hyperparathyroidism of renal origin that are unique to patients with CKD [Mathew et al.
Atherosclerosis can also be linked to vascular diseases in the eye. Retinal arteries may become blocked when a blood clot or fat deposits get stuck in the arteries. These blockages are more likely if there is atherosclerosis in the eye [Medical Encyclopedia, Retinal artery occlusion. Retrieved from https://medlineplus.gov/ency/article/001028.htm.]. If the central retinal artery is affected, i.e. the artery is narrowed, the blood supply to the inner retina can be reduced, which can cause loss of vision. Vascular hypertension can also occur in association with atherosclerosis, which can lead to vascular occlusions of the retina.
Atherosclerosis can also play a role in neurodegenerative diseases, such as Alzheimer's disease. According to Roher et al. [Roher et al. 2003] atherosclerosis-induced brain hypoperfusion contributes to the clinical and pathological manifestations of Alzheimer's disease. Alzheimer's disease is a progressive brain disorder, that causes a loss of brain cells that leads to memory loss and the decline of other thinking skills.
Diseases Associated with H2S
H2S was shown to exert various effects on the different systems, like the gastrointestinal, neuronal, cardiovascular, respiratory, renal, and hepatic systems [Singh, S. B., & Lin, H. C. 2015]. Hydrogen sulfide reduces arterial blood pressure, limits atheromatous plaque formation, and promotes vascularization of ischemic tissues. It has been shown that H2S is a proangiogenic substance, which can restore ischemic tissue function, and it has a beneficial effect in treating cerebral artery occlusion, and post-ischemic cardiac remodelling, as shown in animal models [Kanagy, N. L., Szabo, C., & Papapetropoulos, A. 2017].
Hydrogen sulfide reduces the amount of reactive oxygen species (ROS) by inhibiting ROS production, by direct scavenging and by increased expression of antioxidant enzymes. Thus, H2S can reduce oxidative stress and thereby counteract oxidative stress-related changes in the vessels, such as hypertension, atherosclerosis, and vascular diabetic complications [Kanagy, supra].
The present inventors have shown that treating ApoE−/− mice with the DHEA-derived steroid S2 according to the invention increases H2S levels in the serum, thus it can be reasonably expected to have beneficial effects in treating not just atherosclerosis-associated diseases, but other oxidative stress-related disorders or pathologies associated with decreased levels of H2S.
According to the review of Zhang et al. (2013) [Zhang et al. 2013] H2S is involved in aging as well, through by inhibiting free-radical reactions. Moreover, it has been shown that H2S has a therapeutic potential in age-associated diseases [Kanagy, supra].
Antioxidant effects of hydrogen sulphide have been demonstrated in the context of atherosclerosis. H2S delayed progression of the disease, and/or reduced its severity.
Atherosclerosis is preferably treated in patients diagnosed with atherosclerosis or preventive treatment is applied in patients at risk of arteriosclerosis or atherosclerosis.
Diagnosis of atherosclerosis includes measurement of a locally decreased blood pressure (near to the site of atherosclerotic lesions). Also changed blood flow may be heard by experts via stethoscope or a weak or absent pulse below the arterial area narrowed due atherosclerotic lesions can also be a sign of atherosclerosis.
Diagnosis of Atherosclerosis May Also Include the Following Diagnostic Tests:
Typically, therapeutic methods include diagnosis and once signs or symptoms of atherosclerosis is/are established therapy may begin.
Atherosclerosis begins decades before the appearance of its clinical consequences. As to the risk of arteriosclerosis and atherosclerosis, it is advisable to start preventive treatment in patients having a significant risk of developing these diseases.
Risk factors typically include obesity, diabetes mellitus type II, elevated low-density lipoprotein (LDL) cholesterol levels, metabolic syndrome and family history of atherosclerosis or associated disease all present, by way of example, increased risk factors. As to life management a sedimentary lifestyle, smoking, cholesterol-rich diet etc. present further risk factors.
While guidelines may differ per countries on diagnosis and may change in time, virtually all guidelines recommend the initial evaluation of individual risk of future cardiovascular disease (CVD) [Libby, 2019]. It is within the skills of a medical doctor to decide on preventive treatment which may be started once risk of development of arteriosclerosis and/or atherosclerosis exists.
Dose of preventive treatment may be lower than those of therapeutic treatment and length of administration may be longer.
In case when atherosclerosis has been manifested and cannot be reversed fully, the treatment may have a therapeutic feature in that an effort is made to reverse, at least partially, the lesions or plaques formed, and a preventive feature to avoid further lesions or plaques to be formed.
In certain cases, in particular if the risk factors support this decision and/or if no full reversal of the symptoms is achieved, the medical doctor responsible for the treatment may decide that long term treatment is necessary. Aging provides an additional risk factor, e.g. in case of patients above 40 years or above 50 years or in particular above 55 years or above 60 years long term treatment may be justified.
Long term treatment may mean administration of the compound or pharmaceutical composition of the invention for at least 6 months, or for at least 1 year, 2 years, 5 years or lifelong.
In the present invention the stereoisomers, enantiomers or diastereomers may be present in the pharmaceutical compositions. In preferred compositions the active isomers are enriched. Various isomers are disclosed hereinabove and in the examples.
Pharmaceutically acceptable salts may be obtained by treating a compound with a corresponding base or salt of the cation to be added to the composition.
The compounds of the invention may exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water. In general, the solvated forms are considered equivalent to the unsolvated forms for the purpose of the invention.
As to pharmaceutically acceptable carriers and excipients, the term acceptable means being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.
For oral administration, the active ingredient may be presented in a form as described in the Brief description of the invention section.
In general, preparation of a composition with a pharmaceutically acceptable carrier or excipient may be carried out as described in e.g. as a standard reference, Gennaro, A. R. et al., Remington: The Science and Practice of Pharmacy (20th Edition., Lippincott Williams & Wilkins, 2000, see especially Part 5: Pharmaceutical Manufacturing).
More specifically, oral lipid-based drug delivery systems can e.g. be prepared as described in the review paper Kalepu, S, Oral lipid-based drug delivery systems—an overview. Acta Pharmaceutica Sinica B, 2013 3 (6) 361-372.
Solubilization of oral and injectable formulation is taught e.g. in Strickley, R. G. Solubilizing Excipients in Oral and Injectable Formulations. Pharmaceutical Research, Vol. 21, No. 2, February 2004.
The pharmaceutical composition of the invention may be presented in unit-dose or multi-dose containers.
In case of oral preparation, solid dosage units, such as pills, tablets, etc. as given above, can be provided.
Injection liquids can be presented e.g. in predetermined amounts, for example in sealed vials and ampoules, and may also be stored in the form of dry crystals or freeze dried (lyophilized) condition requiring only the addition of sterile liquid carrier, e.g. water or physiological salt solution, prior to use.
The compounds of the present invention are present in the pharmaceutical compositions in a purity appropriate for human or animal administration. Purification techniques include e.g. recrystallization as shown in the examples and/or chromatographic purification as well known in the art.
The present invention is further illustrated by way of non-limiting examples.
All reagents were purchased from Sigma-Aldrich (Darmstadt, Germany) with ACS grade or higher purity, exceptions are specified below including DHEA. Steroid compounds were taken up in tap-water followed by sonication. DHEA-derived steroid was produced by the inventors as described in Examples. The reagents used to synthesis were from Reanal, Budapest, Hungary (“alt” quality).
All the animal experiments were approved by the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Animal experiments performed in this study were approved by the Scientific and Research Ethics Committee of the Scientific Council of Health of the Hungarian Government under the registration number of DE MÁB/157-5/2010 and are reported in accordance with the ARRIVE guidelines. C57BL/6 ApoE−/− mice (The Jackson Laboratory; B6.129P2 Apoetm1Unc/J; RRID: IMSR_JAX: 002052) were maintained at the University of Debrecen under specific pathogen-free conditions in accordance with guidelines from Institutional Ethical Committee. To induce atherosclerotic plaque formation, standard chow diet was changed to atherogenic diet (15% fat, 1.25% cholesterol, ssniff Spezialdiäten GmbH, Soest, Germany) at the age of 8 weeks. Mice were randomly divided into two groups and parallelly with the atherogenic diet, mice received tap-water or DHEA-derived steroid (100 μg steroid/mouse/day) in tap-water. The DHEA-derived steroid was replaced every day. Aortas were harvested after 8 weeks of treatment. All mice were euthanized by a predictable and controllable administering of slow-fill compressed CO2 asphyxiation. Atherogenic food composition (high-fat diet): Crude Nutrients (%): Crude Protein 19%; crude fat 15.2%; crude fiber 3.4%; crude ash 6.3%; starch 25.6%; sugar 11.2%; Additives (per kg): vitamin A 15,000 IU; vitamin D3 1,000 IU; vitamin E 110 mg; vitamin K3 5 mg; vitamin C 0 mg; copper 13 mg.
Aortas were dissected from the aortic arch to the iliac bifurcation. After that, the fatty tissue was carefully removed under a stereomicroscope (M125; Leica Biosystems Nussloch GmbH). The atherosclerotic lesions were revealed by en face Oil Red O (Sigma-Aldrich, Lot. 00625;) staining. 0.07 g ORO powder was dissolved in 25 mL methanol and mixed for 10 min on a magnetic stirrer. Finally, 10 mL 1 mol/L NaOH was added to the ORO solution and filtered it. Images were taken with the camera (Nikon D3200; Nikon Corp., Japan) and whole aorta plaque area was analyzed with ImageJ 1.49 software.
Immunohistochemistry from the aortic root was performed on formalin-fixed, paraffin-embedded tissue sections. 4 μm slides were then deparaffinated using xylol and ethanol. The serial sections were stained with Gill Hematoxylin solution (Merck) followed by eosin counterstaining or with von Kossa. For digital documentation, the stained specimens were scanned with a Mirax Midi scanner (3D Histech, Budapest, Hungary). Macroscopic pictures of the arteries were taken with Nikon D3200 camera (Nikon Corp.; Minato, Tokyo, Japan).
Determination of Sulfide Level from Tissue with Zinc Precipitation Assay
Sulfide levels were measured with zinc precipitation method based upon developed by Gilboa-Garber [Gilboa-Garber N. 1971] and modified by A. D. Ang et al. [Ang A D, et al. 2012]. The human carotid artery was homogenized under liquid nitrogen in 7.4 pH PBS and sonicated it. After sonication the sample was centrifuged at 12000 G for 15 min and the lipids free clear supernatant was collected. 200 μL sample was mixed with 350 μL 1% zinc acetate and 50 μL 1.5 mol/L sodium hydroxide and incubated for 60 minutes on a shaker. Incubation step was followed by centrifugation at 2000 G for 5 minutes to pellet the generated zinc sulfide. The supernatant was then removed, and the pellet washed with 1 mL of distilled water by vortexing extensively, followed by centrifugation at 2000 G for 5 minutes. The supernatant was then aspirated off and the pellet reconstituted with 160 μL of distilled water and mixed with 40 μL of pre-mixed dye (20 μL of 20 mmol/L dimethyl-p-phenylenediamine dihydrochloride (NNDP) in 7.2 mol/L hydrochloric acid (HCl) and 20 μl of 30 mmol/L Iron (III) chloride (FeCl3) in 1.2 mol/L HCl). After 10 min the absorbance of the generated methylene blue (MB) was measured with a spectrophotometer at 667 nm. Since during the reaction 1 mol/L MB formed from 1 mol/L sulfide, the concentration was determined by the MB's extinction coefficient (30 200 M 1 cm 1). Samples were normalized for protein concentration.
“N” represents the number of tissue samples used in each group. The “n” denotes the number of replications of the independent results.
Data were analyzed by GraphPad Prism 5.02 software (GraphPad Software Inc., 7825 Fay Avenue, Suite 230 La Jolla, CA 92037; RRID: SCR_002798). All statistical data are expressed as mean±SEM. If data groups passed the normality test and equal variance test, we performed Student's t-test or One Way ANOVA followed by Bonferroni post hoc tests as indicated in the Brief description of the figures. P<0.05 was considered significant.
DHEAS can be quantitatively determined in biological samples as described e.g. by Sánchez-Guijo et al. [Sánchez-Guijo et al., 2015] and papers referenced by them.
Name of the compounds prepared:
Preparation of 17-lauroyloxy-5-androstene-3-sulphate sodium salt (compound S2)
3-hydroxy-17-oxo-5-androstene (DHEA) (10)
3-acetoxy-17-oxo-5-androstene (1)
3-acetoxy-17-hydroxy-5-androstene (2)
3-acetoxy-17-hydroxy-5-androstene (3)
3-hydroxy-17-lauroyloxy-5-androstene (4)
17-lauroyloxy-5-androstene-3-sulphate sodium salt (5)
1. 3-acetoxy-17-oxo-5-androstene (1)
Into a round bottom flask with magnetic stirrer 25 cm3 dichloromethane (DCM) and 5.8 g (20 mmol) DHEA was measured and added. The solution was cooled to T=0° C. and a mixture of 10 cm3 dichloromethane, 3.1 g acetic anhydride and 200 μl conc. H2SO4 was added drop by drop. The mixture was stirred for 3 hours (t=3 hours) then poured into 50 cm3 5% (w/v) NaHCO3 solution (T=5° C.) and stirred for 10 min; thereafter further 50 cm3 DCM was added. The organic phase was washed with water then with 20% (w/v) NaCl solution and dried over Na2CO3. The solvent was removed in vacuum (Rotadest). The product was stirred with a mixture of 50 cm3 methanol and 50 cm3 water for 15 min, then cooled to T=0° C.
The white crystals of 3-acetoxy-17-oxo-5-androstene (compound I) were filtered out and washed with a small amount of cold solvent then with water and then dried in a vacuum desiccator at T=50° C.
Yield: =5.2 g; R=78.8%
Compound I (4.96 g) and 180 cm3 methanol was added to a flask with magnetic stirrer and cooled to T=−5° C. To this cooled solution 0.7 g sodium-borohydride was added in multiple parts during about 30 min. Thereafter the mixture was allowed to react for a further 2.5 hours and then the reaction mixture was mixed with 100 cm3 0.4 M cold hydrochloric acid (HCl). The mixture so obtained was neutralized to pH 6 by 1 M NaHCO; (about 25 cm3) and diluted by further 50 cm3 water. The product was filtered out, then washed with 2×15 cm3 water: methanol (1:1) mixture and with 2×25 cm3 water.
After vacuum drying (T=50° C.) 4.1 g product was obtained (R=82.0%).
Into a flask with magnetic stirrer, 20 cm3 DCM, 15 cm3 pyridine, 3.35 g (10 mmol) 3-acetoxy-17-hydroxy-5-androstene (compound II) were measured and added, then at T=20-25° C., during 20-30 min, a mixture of 3.1 g (14 mmol) lauric acid chloride (lauroyl chloride) and 10 cm3 DCM was added.
Thereafter the mixture was allowed to react for an additional 5.0 hours (t=5.0 hours), then the mixture was diluted by 50 cm3 DCM and mixed with 50 cm3 10% hydrochloric acid at T=5-10° C. The separated organic phase was washed twice with 10% hydrochloric acid, dried by sodium sulphate, and then the solvent was removed in a rotary evaporator (Rotadest) at reduced pressure in water-bath at T=55° C.
The crude product was suspended by 40 cm3 water: MeOH (1:1) mixture at T=55-60° C., then cooled to T=5° C., and then the crystals were filtered out and washed with 2×10 cm3 of the aforementioned solvent.
After vacuum drying (T=50° C.), 4.3 g of compound III was obtained (R=83.4%).
3.1 g (6.0 mmol) of compound III obtained in the previous step was dissolved in 120 cm3 methanol at room temperature, then 5.4 cm3 1 M NaOH (5.4 mmol) was added, and the mixture was stirred for 12 hours. Then 60 cm3 water was added to the mixture and the reaction mixture was acidified to pH 6 by 2 M hydrochloric acid. The precipitated product was filtered out and washed with 20 cm3 water: MeOH (1:1) mixture and then with 2×20 cm3 water. The wet material was dissolved in 50 cm3 chloroform, extracted (by shaking) with 2×20 cm3 water, and then the solution was dried by Na2SO4, and then the solvent was removed in Rotadest. The product was dried in vacuum dryer (T=50° C.). 2.30 g of compound IV was obtained (R=80.0%).
Into a flask with magnetic stirrer, 25 cm3 DCM, 10 cm3 pyridine and 1.42 g (3 mmol) 3-hydroxy-17-lauroyloxy-5-androstene (compound IV) were measured and added. The reaction mixture was cooled to T=−5° C., and then 0.420 g (250 μl) chlorosulfonic acid (sulfurochloridic acid) mixed with 5 cm3 cool dichloromethane was added to the mixture. After allowing to react for 4.0 hours, 20 cm3 5% sodium bicarbonate solution and 20 cm3 water was added to the mixture at room temperature, then the mixture was stirred vigorously for 30 min.
The three-phase reaction mixture was diluted by 50 cm3 diisopropyl ether and after 15 min stirring the mixture was filtered. The filtered out crude product was dried in vacuum dryer (T=50° C.), then stirred for 30 min at T=50-55° C. After cooling it down, the target product was filtered out, washed with 2×10 cm3 water, and then again dried in vacuum.
The dry product was suspended by 50 cm3 DCM at T=40° C., and after cooling was filtered out and on the filter it was washed again twice with 15 cm3 DCM. Again, it was dried in a vacuum dryer. 1.1 g product was obtained, R=63.9%.
NMR spectrum of the compound is shown in
The other two related compounds (compounds C6 and C16) were also obtained in the outlined reaction pathway. In these cases, at first the acylation of intermediate II with hexanoyl chloride or palmitoyl chloride, respectively, was carried out.
Molecular formula: C27H42O4 MW: 430.6
Into a flask with magnetic stirrer, 25 cm3 DCM (or chloroform), 20 cm3 pyridine and 4.0 g (12 mmol) 3-acetoxy-17-hydroxy (II) intermediate were measured and added. After that, during cooling it by cool water, 2.16 g (16 mmol) hexanoyl chloride mixed with 10 cm3 solvent was added drop by drop at T=20° C., during about 30 min.
After allowing the mixture to react for 3 hours, the reaction mixture was diluted by 50 cm3 solvent and was added to V=150 cm3 10% hydrochloric acid aqueous solution at T=5-10° C. during about 30 min (vigorous stirring!), and then after stirring for 15 min the phases were separated. The organic phase was washed twice with water and once with 10% saline, then dried by sodium sulphate, and then the solvent was removed in Rotadest at reduced pressure.
The product was suspended by 50 cm3 water: MeOH (1:1) mixture at T ˜ 55° C., then cooled down to T ˜ 5° C. and was filtered. The filter cake of crystals was further washed with 2×15 cm3 solvent.
After vacuum drying (T=50° C.), 4.1 g product was obtained (R=79.3%).
Molecular formula: C25H40O3 MW: 388.5
Into a flask with magnetic stirrer, 200 cm3 methanol and 3.9 g (9 mmol) of diacyl derivative prepared in the previous step were measured and added at T=22° C. Then during about 30 min with vigorous stirring 8.2 cm3 c=1M sodium hydroxide solution was added drop by drop. After stirring for 4 hours, 100 cm3 water and 20 cm3 c=0.4M HCl solution were added to the reaction mixture. After stirring for 15 min, pH was adjusted to pH=6.5-7.0 by 5% NaHCO3.
The product was filtered out and was washed with 2×25 cm3 water: MeOH (1:1) mixture and then with 25 cm3 water.
The wet material was dissolved in 50 cm3 chloroform and extracted by shaking with 2×25 cm3 water, then dried by sodium sulphate, and then the solvent was removed in Rotadest at reduced pressure and in water-bath at T ˜ 55° C. Finally, it was dried in vacuum dryer (T=50° C.). 2.6 g product was obtained (R=74.2%).
Into a flask with magnetic stirrer, to a solution of 2.34 g (6 mmol) 3-hydroxy-17-hexanoyloxy-5-androstene dissolved in chloroform (25 cm3), 2.5 cm3 pyridine was added. The reaction mixture was cooled down to T=0-(−5° C.) and during about 20-30 min a solution of 880 mg chlorosulfuric acid and T<0° C. chloroform was added drop by drop.
Thereafter, the mixture was allowed to react for an additional 4.0 hours at T ˜ 5° C., and then 35 cm3 5% sodium bicarbonate solution and 20 cm3 water, and 50 cm3 diisopropyl ether were slowly added. The heterogeneous reaction mixture was stirred for 15 min, and the solid product was filtered out and then dried in vacuum dryer (T=50° C.). Then the material was stirred with 20 cm3 water for 30 min at T=50-55° C.
After cooling it down, the target product was filtered out and washed with 2×10 cm3 water, and then again dried in vacuum dryer.
The dry crystallic material was stirred with 50 cm3 DCM at T=40° C. for 30 min, then after cooling it down was filtered out and washed with 2×15 cm3 solvent, and then again dried.
1.8 g of 17-hexanoyloxy-5-androstene-3-sulphate sodium salt was obtained (R=61.2%).
NMR spectrum of the compound is shown in
Molecular formula: C37H62O4 MW: 570.8
In a flask with magnetic stirrer, in a mixture of 15 cm3 chloroform and 10 cm3 pyridine 3.0 g (9 mmol) 3-acetoxy-17-hydroxy-5-androstene was dissolved, and during cooling down, in about 30 min, at T=20° C., a mixture of 10 cm3 chloroform and 3.3 g (12 mmol) palmitoyl chloride was added.
After stirring for an additional 5.0 hours, the reaction mixture was diluted by 25 cm3 chloroform, and then it was added to V=100 cm3 10% hydrochloric acid solution at T=5-10° C., during vigorous stirring. Then the separated organic phase was washed twice with water and then was extracted by shaking once with 10% sodium chloride solution. The mixture was dried by sodium sulphate and the solvent was removed in Rotadest at reduced pressure (water-bath, T ˜ 50° C.).
The crystallic product was suspended by 50 cm3 water: MeOH (1:1) mixture, then cooled down to T<10° C., and then it was filtered out and washed with 2×10 cm3 solvent.
After vacuum drying (T=50° C.), 3.9 g product was obtained (R=75.9%).
Molecular formula: C35H60O3 MW: 528.7
Into 300 cm3 methanol, 3.42 g (6.0 mmol) diacyl compound was measured and added, and then at first the suspension was heated to T=55° C., and then swiftly cooled down to T ˜ 20-22° C. After that, during about 30 min, a mixture of 5.4 cm3 c=1.0 M sodium hydroxide and 10 cm3 methanol was added drop by drop.
The reaction mixture was stirred for 36.0 hours at room temperature. During this long period, 85-90% conversion can be reached. Allowing to react for an additional time is inadvisable as it would produce unwanted by-product, and the remaining ˜15% starting material does not hamper the final sulphation reaction.
After the hydrolysis, 100 cm3 water and 20 cm3 c=0.4 M hydrochloric acid were added to the reaction mixture. Then, about 80% of the methanol was distilled in Rotadest at reduced pressure in water-bath at T ˜ 55° C. The suspension was cooled down, filtered, and the solid product was washed with 2×20 cm3 water: MeOH (1:1) mixture and then with 2×20 cm3 water.
After vacuum drying, 3.0 g, ˜ 85% product was obtained (˜2.55 g active substance; R: ˜ 80%).
Into the usual equipment, 15 cm3 chloroform, 2.0 cm3 pyridine and 2.4 g (˜4 mmol) 17-hydroxy derivative were measured and added. The mixture was cooled down to T=0-(−5° C.), and then during 20-30 min a mixture of 10 cm3 T ˜ 0° C. chloroform and 590 mg chlorosulfuric acid was added drop by drop.
After that, the mixture was stirred for 5 hours at T ˜ 5° C., and then 25 cm3 5% sodium bicarbonate solution and 20 cm3 water, and 40 cm3 diisopropyl ether were added slowly. The three-phase mixture was stirred for 15 min, and then the product was filtered out and then dried in vacuum dryer (T=50° C.).
After that, the crude product was stirred with 20 cm3 water at T=50-55° C. for 30 min, and after cooling it down, the product was filtered out and washed with 2×10 cm3 water. The product was dried in vacuum dryer (T=50° C.). The dry crystallic material was stirred with 50 cm3 DCM at T=40° C. for 30 min, then cooled down and then was filtered out and washed with 2×15 cm3 solvent.
After vacuum drying, 1.5 g of 17-palmitoyloxy-5-androstene-3-sulphate sodium salt was obtained (R=59%).
NMR spectrum of the compound is shown in
In order to investigate, whether DHEA-derived steroid has an anti-atherogenic effect, the present inventors performed experiments with an atherosclerotic animal model. DHEA-derived steroid drinking mice were sacrificed after 8 weeks and aortas were dissected from aortic arch to iliac bifurcation. The formation of atherosclerotic lesion was revealed by lipid staining. As en face Oil Red O staining of the whole aorta presented by
ApoE−/− mice on a high-fat diet exhibited an expansion of extracellular matrix deposition in aortic valves. As demonstrated by von Kossa staining, calcific nodules appearing in the aortic heart valves of ApoE−/− mice have been provoked by high-fat diet (
The present inventors' research group recently revealed, that H2S has a strong ability to inhibit atherosclerotic plaque formation and calcification via attenuation inflammation, as well as prevention LDL oxidation, scavenging reactive oxygen species [Potor L, et al. 2018; Sikura K E, et al. 2019]; and reversing aging-associated pathologies [Perridon B W, et al. 2016; Zhan J-Q, et al. 2018; Hou C L, et al. Thus, the present inventors measured the H2S presence in the mice plasma derived from tap-water drinking or DHEA-derived steroid treated ApoE−/− mice. They found that DHEA-derived steroid treated mice plasma's H2S concentration was approximately 2.4 times higher compared to the control mice plasma (
To investigate the specific inhibitor effects of the S2 steroid in atherosclerosis, the present inventors performed animal experiments with the basic molecule of S2 steroid derived from DHEA. Additionally, to further investigate the characteristic of S2 the present inventors examined the sulfide side chain of S2 using Dehydroepiandrosterone sulfate (DHEA-S). Finally, the present inventors also investigated the length of carbon side chains of S2 in atherosclerosis using 17-hexanoyloxy-5-androsten-3-sulfate sodium salt (C6) and 17-palmitoyloxy-5-androsten-3-sulfate sodium salt (C16) steroid. The present inventors found that, neither DHEA (N=11), DHEA-S(N=7), C6 (N=3) and nor C16 (N=3) molecules can abrogate atherosclerotic plaque formation in mice (
The present inventors investigated the DHEA and DHEA-S effects on calcification of mouse heart valves. The present inventors found that neither DHEA nor DHEA-S is able to inhibit mineralization of aortic valve compared to S2 steroid (
From a pharmacological point of view to the best of inventors' knowledge, there is no medicine in the market to reverse atherosclerosis and calcification of vessels. Current drugs only slow down the progression of atherosclerosis. Therefore, the present inventors examined the possibility of S2 steroid treatment to reverse the already formed atherosclerotic lesion and calcification. In this experiment the present inventors kept the mice on atherogenic diet for 4 or 6 weeks for development of atherosclerotic lesion and calcification. After that the present inventors changed the atherogenic diet to chop diet and they added S2 steroid to the mice's water for 4 weeks and 8 weeks. Control group got tap water only. Surprisingly the present inventors observed significantly lesser atherosclerotic lesion in case of S2 therapy (
Despite the improving tendency in the past few years, diseases developing on the basis of atherosclerosis (myocardial infarct, stroke, obliterating lower extremity artery disease, Alzheimer's disease, circulation problems of the eye) still lead the morbidity and mortality statistics worldwide. Considering that billions of people are affected by these diseases, after a possibly successful medical innovation of the new compound of the present inventors, it could be a drug used both preventively and therapeutically without endocrinological side effect. The human, scientific, professional, and not least of all economic impact is hardly evaluable.
The present study demonstrates that DHEA-derived S2 steroid represents a novel atheroprotective drug, usable for preventing the formation of lipid derivatives in the atherosclerotic plaque and inhibits calcification of heart valves. Based on the present study, DHEA-derived steroid (S2) induced elevated H2S (I.) scavenged atherogenic free radical superoxide. (II.) limited the formation of pro-oxidant and pro-inflammatory lipid mediators and subsequent endothelial responses provoked by these species. (III.)
prevented heart valves calcification by controlling osteoblast-like differentiation of valvular interstitial cells.
In conclusion, the present study provides evidence that DHEA-derived steroid is a potent preventive and therapeutic agent in atherosclerosis and aortic valve calcification.
Mathew, R. O., Bangalore, S., Lavelle, M. P., Pellikka, P. A., Sidhu, M. S., Boden, W. E., and Asif, A. (2017). Diagnosis and management of atherosclerotic cardiovascular disease in chronic kidney disease: a review. Kidney International, 91 (4), 797-807.
| Number | Date | Country | Kind |
|---|---|---|---|
| P2200041 | Feb 2022 | HU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/HU2023/050005 | 2/17/2023 | WO |
